Immunotherapy is very promising for the treatment of patients with cancer. The numerous clinical protocols carried out which have used therapies based on cytokines, infusions of effector T cells or vaccination protocols have demonstrated that cancer immunotherapy is generally safe. However, although the induction of immune response after the treatment has been observed in these clinical protocols, most of the patients are incapable of developing an effective antitumor response. The demonstration of the presence of Treg lymphocytes in the tumor tissue or the lymph nodes of patients with melanoma, lung cancer, ovarian cancer, pancreatic cancer and breast cancer as well as in hepatocarcinomas (Nishikawa H. et al., “Regulatory T cells in tumor immunity”, Int. J. Cancer, 2010, vol. 127, pages 759-767) and the description that tumor tissue secretes chemokines which specifically attract this subpopulation towards tumor tissue, indicate that the access of Treg lymphocytes to the tumor is a dynamic process and that it exerts an immunosuppressive effect facilitating the progression of the disease.
The regulatory T cells (Treg cells or Tregs), formerly known as suppressor T cells, are a subpopulation of T cells which modulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune disease. Treg cells are immunosuppressive and generally suppress or downregulate induction and proliferation of effector T cells. Treg cells express the biomarkers CD4, FOXP3, and CD25 and are thought to be derived from the same lineage as naïve CD4 cells.
The presence of Treg in the tumor as well as in peripheral nodes could explain the low efficacy of the immunotherapy protocols. In the same way, in infectious diseases, the control exerted by Treg lymphocytes can limit the magnitude of the effector T responses and cause the failure in the control of the infection. It has thus been described that some viruses such as hepatitis B virus, hepatitis C virus and HIV (Joosten S. A. et al., “Human CD4 and CD8 regulatory T cells in infectious diseases and vaccination”, Hum. Immunol., 2008, vol. 69(11), pages 760-70) can use Treg lymphocytes to block the antiviral immune response and thus allow the establishment of the persistent chronic infection. Due to all this, it is believed that the modulation of the action of Treg lymphocytes can be essential in the development of immunotherapies against cancer or against infectious diseases.
In this regard, methods for inhibiting the activity of Treg lymphocytes have been disclosed in the prior art, in an attempt to regulate their negative effect on the immune system. Some of these methods involve the elimination of Treg cells, by means of using depleting antibodies or by means of blocking the cytokines that they produce and which may be responsible for their activities (TGF-β, IL-10). The methods which are based on the depletion of the regulatory T cells have the drawback that they eliminate the cells and involve risks of causing autoimmune diseases.
In an attempt to find alternative immunotherapies, FOXP3 (forkhead box P3), also known as scurfin, attracted the interest of the scientists. This protein is member of the FOX protein family, and appears to function as a master regulator of the regulatory pathway in the development and function of regulatory T cells. Moreover, in addition to naturally occurring Treg which are generated in the thymus, FoxP3 expression can be induced in the periphery in CD4+CD25− T cells through TCR crosslinking, leading to attenuation of effector functions in the stimulated cells (proliferation and cytokine production) (Reviewed in Lozano T. et al., “Searching for the Achilles Heel of FOXP3”, Front. Oncol., 2013, vol. 3, page 294). The immune-suppressive tumour microenvironment, affects antigen presentation to tumor-specific T cells and may result in suboptimal T-cell activation and T-cell tolerance. In this regard, expression of FoxP3 after suboptimal TCR stimulation of CD4+ in the presence of immunosuppressive cytokines TGF-β, IL-6 or IL-10 and other metabolites may have an important role governing the functionality of transferred lymphocytes favouring T-cell-tolerization. It has become evident that Foxp3 can be transiently expressed in activated human or murine CD4+ T cells acquiring some features of Treg cells (Reviewed in Lozano T. et al., 2013, supra). But, such induced FoxP3 expression may not be restricted to CD4 T cells. Indeed there are increasing reports on the existence/induction of CD8+FoxP3+ T cells in cancer and chronic infections (Frassanito M. A. et al. “Myeloma cells act as tolerogenic antigen-presenting cells and induce regulatory T cells in vitro”, Eur J Haematol., 2015, vol. 95(1), pages 65-74). These findings suggest that FoxP3 may serve to shut off T cell activation, acting as a broad regulator of immune response, and thus, FoxP3 can be considered as a potential therapeutic target.
The molecular basis of FOXP3 function has been poorly understood. It has been described that the transcription factor scurfin (FOXP3, expression product of the foxp3 gene) (Williams L. M. and Rudensky A. Y., “Maintenance of the Foxp3-dependent developmental program in mature regulatory T cells requires continued expression of Foxp3”, Nat. Immunol., 2007, vol. 8, pages 277-84) is essential for the activity of Treg lymphocytes, such that its presence determines the suppressive activity of these cells. The cDNA sequences encoding murine and human scurfin have been the object of U.S. Pat. No. 6,414,129 which furthermore describes that the modulation of the expression of scurfin can have therapeutic effects in various diseases; said patent also mentions the use of synthetic peptides, among other molecules, to regulate the expression of the foxp3 gene but does not mention anything about the possibility of inhibiting the activity of the already expressed scurfin.
FOXP3 capacity to bind DNA is critical for its functionality and it is known that FOXP3-DNA interactions are assisted by other cofactors and by multimerization. Growing numbers of transcription factors that interact with FOXP3 are being identified and some have been implicated in the Treg cell-specific gene expression program (Reviewed in Lozano T. et al., 2013, supra). FOXP3 has various distinguishable functional domains: (i) a N-terminal domain (from aa 1 to 193, with two proline-rich regions), (ii) a zinc finger (aa 200-223) and a leucine zipper-like motif (aa 240-261) (ZL domain) located in the centre of the protein and (iii) the highly conserved carboxy terminal forkhead domain (FKH; from aa 338 to 421) responsible for binding to DNA. It has been described that the intermediate region is implicated in FOXP3 dimerization, which is required for its function as a transcriptional regulator (Reviewed in Lozano T. et al., 2013, supra). Also, the physical interaction of this region with the transcription factor AML1 (acute myeloid leukaemia 1)/Runx1 (Runt-related transcription factor 1), suppresses IL-2 and IFN-γ production, upregulates Treg-associated molecules, controls anergy of the cell and exerts Treg suppressive activity (Reviewed in Lozano T. et al., 2013, supra) Thus, those strategies able to inhibit FOXP3 dimerization, its interaction with AML1 or to modify the FOXP3 interactome might have important consequences on Treg activity and thus could be exploited as therapeutic agents in cancer.
In a previous work, it was identified the 15-mer synthetic peptide of sequence SEQ ID NO: 1 (hereinafter also referred “p60”)
(SEQ ID NO: 1)ArgAspPheGlnSerPheArgLysMetTrpProPhePheAlaMetwhich entered the cells, bound to FOXP3 and inhibited murine and human-derived Treg, improving effector T-cell stimulation in vitro and in vivo (Casares N. et al., “A peptide inhibitor of FOXP3 impairs regulatory T cell activity and improves vaccine efficacy in mice”, J. Immunol., 2010, vol. 185(9), pages 5150-5159).
In spite of the efforts made in the field of immunotherapy, there is still the need of providing compounds with an improved efficiency in regulating or blocking Treg cells.